Random mutagenesis using a mutator strain

Random mutagenesis using a mutator strain

This protocol has been confirmed to work for the following organisms.

• Streptomyces coelicolor

Purpose

Mutator strains are useful for generating random mutations in your gene of interest. A library of random mutants is generated in an Escherichia coli mutator strain, pooled and transformed into ET12567/pUZ8002 (or another suitable strain) for intergeneric conjugation into your Streptomyces. The standard ET Conjugation protocol is then used to transfer the library of mutant vectors into your desired Streptomyces strain. A suitable genetic screen or selection is then used to identify mutant(s) of interest.

Method

Materials needed

Strains needed:

1. E. coli XL-1 Red competent cells (Tet^R; Agilent))
2. E. coli ET12567/pUZ8002 (Km^R, Chl^R)
3. Streptomyces strain of your choice

Other materials:

1. Your chosen vector for mutagenesis
2. Liquid LB medium
3. Liquid SOC medium
4. Antibiotic stock solutions (See the Antibiotic stocks and working concentrations page for recommended antibiotic concentrations.)
5. Solid LB agar containing antibiotic to select for your vector of choice
6. Solid LB agar containing antibiotic to select for your vector of choice, kanamycin, and chloramphenicol (optional)
7. Miniprep kit
8. Electroporator
9. Sterile loop/toothpicks, spreader, Eppendorf tubes, pipet tips, etc.

Protocol

• Transform your vector into XL-1 Red competent cells (Agilent) following the manufacturer's protocol and plate on LB medium containing the appropriate antibiotic to select for your vector. Incubate plate(s) overnight at 37ºC.

• Using a sterile loop or toothpick, pick a large number of transformants (~200 or more colonies) into liquid LB medium containing the appropriate antibiotic to select for your vector, and incubate overnight with shaking (250 rpm) at 37ºC.

• After overnight incubation, make a miniprep of your now-mutated vector. If desired, you may also sub-culture the culture to
• This new E. coli ET12567 strain, containing both pR9604 and your desired kanamycin-marked vector can now be glycerol stocked, and used to conjugate your vector into Streptomyces via the standard ET Conjugation protocol.

Notes

1. Ampicillin and Carbenicillin can be used interchangeably.
2. The E. coli strain ET12567 contains a chloramphenicol resistance gene integrated into the genome. This marker does not need to be selected for during routine culture of the strain.
3. The XL1-Red strain is resistant to tetracycline, but this marker may be lost at a relatively high frequency due to frequent mutations arising in this strain.

Protocol adapted for ActinoBase by Morgan Anne Feeney from the University of Strathclyde.